The long term goal of this research is to understand the mechanisms of antiarrhythmic drug modification of the cardiac sodium channel. The experimental approach in this grant proposal combines the measurement of Na channel gating current, which are a direct measure of the channel's molecular conformational states, with modification of cardiac Na channels by toxins or agents to help understand antiarrhythmic drug interactions with the cardiac Na channel. In our present grant period we have found the applying this approach has made important advances in the understanding of antiarrhythmic drug modification of cardiac Na channels by QX-222 and lidocaine, and int he understanding of cardiac Na channel inactivation by the use of the site-3 toxin, Anthopleurin-A. For the next grant period we propose to continue our studies of cardiac Na inactivation, which has been shown to be important in the action of antiarrhythmic drugs, and of lidocaine's modification of cardiac Na channel gating currents. This grant proposal will use both native cardiac Na channels in single heart cell preparations and mutated cardiac Na channels expressed in large, fused tsA201 cells to study Na channels modified by lidocaine, by Na channel toxins and agents, and by site-directed mutagenesis. The specific aims are; 1. Determine if batrachotoxin (BTX) modification of cardiac Na channels alters movement of the channels's voltage sensors similar to changes caused by the antiarrhythmic drug, lidocaine, and determine if either lidocaine or BTX inhibit movement of the voltage-sensor associated with cardiac Na channel inactivation from the open-state (OyieldsI inactivation). 2. Determine if the voltage-sensor associated with O yields I inactivation in cardiac Na channels is separate from the putative inactivation gate formed by the intracellular linker region between domains III and IV. 3. Determine if immobilization of off-gating charge in cardiac Na channels is associated with the putative inactivation gate. 4. Determine if the voltage-sensor for O yieldsI activation in cardiac Na channels is associated with the S4 segment of domain IV. 5. Determine if the cardiac Na channel gating charge-voltage relationship measured during step depolarizations to test potentials near INa threshold underestimates the total gating charge. The results of the proposed studies in the this grant should advance our understanding of the molecular mechanisms of cardiac Na channel inactivation, and our understanding of which components of the cardiac Na channel's voltage sensors are modified by lidocaine. In combination, they should contribute to the formation of a better molecular model of antiarrhythmic drug interactions with the cardiac Na channel, and form the foundation for future studies of antiarrhythmic drug-Na channel interactions.